This invention relates to cytological assays and more particularly to a process for detecting and measuring cellular response to certain extracellular substances.
It has long been known that the interior of animal and plant cells is electrically negative with respect to the exterior. The magnitude of this potential difference is generally between 5 and 90 mV, with most of the potential being developed across the cell membrane.
Cell membrane potentials change in several ways with the physiologic state of the cell. Since the expenditure of metabolic energy is required to maintain potentials, the potential across the membrane of an injured or dying cell is decreased in magnitude. More specific changes in membrane potential occur within minutes following the interaction of cells with a wide variety of substances or ligands which bind with relatively high affinity to specific transmembrane receptors.
In relation to ligands and receptors, recent advances in cellular biology have demonstrated that a basic form of communication between the cells of multicellular living systems is effected through extracellular ligands or chemical messengers. These chemical messengers originate both from specialized tissues within the organism and from external sources. Their chemical structures and sites of action vary widely. They can have a zone of influence as small as a few hundred angstroms (e.g. the neuromuscular junction) or throughout the whole organism (blood-borne messengers such as hormones).
As a result of recent biochemical research in this area, it is now established that many of these chemical messengers interface with the cell receiving the message by selectively coupling to receptors located on the exterior of the cell. Apparently, the receptor recognizes the chemical messenger on the basis of its stereochemistry and/or the spatial distribution of its charged or chemically active groups. The ligand and receptor thus become non-covalently bonded in a "lock-and-key" relationship. As a result of binding, an intracellular biochemical or biophysical change takes place which induces the cell to begin or terminate some metabolic or physiological process. Ligands include substances such acetylcholine, epinephrine, and norepinephrine (neurotransmitters), insulin, growth hormone, and thyrotropin (hormones), histamine, antigens, proteins of the immune system or portions thereof, viruses, bacteria, certain toxins, and sperm. For many of these substances there exists a natural or synthetic antagonist, i.e., a substance which can reverse the physiological effect of its corresponding substance, or binds to receptors thereby excluding binding of the natural substance. Natural or synthetic agonists are also known. An agonist is a substance which binds to the receptor to initiate a physiological cellular response similar to that of the natural ligand. Many drugs now commonly employed in the practice of medicine are agonists or antagonists of natural ligands.
In many cases, the events which occur in cells subsequent to the binding of ligand to specific receptors can be duplicated by reacting the cells with other substances which bind to the receptors, e.g., with certain plant lectins or with antibody prepared against isolated receptors. It is also possible to initiate cell responses of this kind by the addition of agents which change membrane potential in the same direction as occurs following ligand-receptor interaction.
A rapid and reliable method of detecting and measuring the intracellular effect of a ligand-receptor interaction would have significant utility in biochemical research and diagnostic testing. Because of the diversity of receptor specificities among otherwise homogeneous cell populations, and because of the enormous number of different chemotactic agents, such a test should ideally be capable of assaying individual cells.
Various direct and indirect methods for detecting ligand-receptor interactions are known in the art. One class of methods employs radioactive or fluorescently tagged ligands. These are incubated with a cell-suspension, and after washing to remove unbound ligands, the radioactivity or fluoresence of the cell mass is assayed. The requirement for tagged ligands places severe limitations on the use of this technique. Another class of prior art detection methods utilizes the observation that ligand-receptor interactions are often accompanied by increases in intracellular deoxyribonucleic acid (DNA) synthesis. In certain systems, following the binding of ligands to receptor, e.g., after lymphocytes have been stimulated with antigen or mitogens such as phytohemagglutinin, DNA levels can be measured and then compared with the DNA level of a comparable cell mass in which no ligand-receptor interaction has occurred. The relative level of DNA provides a measure of the ligand-receptor interaction. This technique is limited in use because the DNA synthesis is generally not detectable for many hours or even days following ligand binding.
Still another class of methods for detecting certain types of ligand receptor interaction is based on changes in the "structuredness of the cytoplasmic matrix" as determined by measurement of the polarization of fluorescence of intracellular fluorescein. In order to be analyzed by such methods, cells must have the ability to produce intracellular fluorescein by enzymatic hydrolysis or fluorescein diacetate or a similar compound. These methods are technically difficult to implement and the results are difficult to interpret. Furthermore, the method requires metabolic modification of a reagent by the cells under study.
While it has been known for decades that excitable cells such as neurons and muscle cells undergo a rapid change in membrane potential when stimulated by a neurotransmitter, it has only recently become apparent that the membrane potential changes induced by physiologic stimuli are not limited to these specialized cells. By inserting microelectrodes into non-excitable cells, researchers have established that membrane potential changes are associated with their ligand-receptor interactions. In fact, it has been observed that the physiological events which occur in non-excitable cells subsequent to the binding of a ligand to a receptor can sometimes be duplicated by the addition to the cell suspension of agents which alter membrane potential in the same direction as occurs following ligand-receptor binding. Further research has developed new techniques for observing such changes. For example, a photometric method of measurement of changes in membrane potential in bulk cell suspensions have been described by P. J. Sims et al. (Biochemistry, Vol. 13, No. 16, 1974, page 3315). According to this method, the cell suspension is incubated with a cyanine or other dye which is positively charged, and capable of traversing the lipid layer of cell membranes. The ratio of intracellular to extracellular dye concentration changes with changes in cell membrane potential: as the cells become hyperpolarized, i.e., as the inside of the cells become more negative, more dye molecules enter the cells. In the Sims et al. method, these dyes are used in concentrations such that intracellular dye molecules form non-fluorescent aggragates, and the fluorescence of the free dye in the suspending medium is measured against the darker background of the cells. This fluorescence decreases with hyperpolarization of the cells. If only a small fraction of the cells in a suspension are sensitive to a given ligand, it is only this fraction which exhibits a change in membrane potential. In this case, the dye concentration in the medium will not change appreciably, and there will thus be no detectable change in the fluorescence of extracellular dye. In any event, this method cannot identify individual cells sensitive to the ligand.
W. N. Ross et al., in the Journal of Membrane Biology, Vol. 33, page 141 (1977) have described the use of merocyanine, oxonol, and cyanine dyes to measure rapid changes of membrane potential in excitable cells such as the giant axons of the squid nervous system. Linear changes in absorption, fluorescence, dichroism, and birefringence of the dyes were found to be associated with changes in membrane potential. The dyes most suitable for these measurements are merocyanines, which are negatively charged and thus do not readily permeate cell walls. Generally, these dyes do not stain cell membranes other than those of excitable nerve and muscle cells. Merocyanines have been observed to stain some non-excitable cell membranes, for example, immature or leukemic blood cells. However, such staining has been shown to occur independently of changes in cell membrane potential.
It is an object of the invention to provide a method of bioassay based on non-intrusive measurement of changes in membrane potential in individual cells. Another object is to provide a rapid, sensitive, and versatile method of bioassay directed to detecting the occurrence of and predicting the cellular response to ligand-receptor interactions. Yet another object is to detect ligand-receptor interactions in individual, non-excitable cells. Other objects are to provide novel techniques for screening potentially active drugs, for diagnosing the physiological malfunctioning of tissues, and for allergy and histocompatability testing. These and other objects and features of the invention will be apparent from the following description of the invention, some preferred embodiments thereof, and from the drawing.